Poxviruses include a large family of DNA viruses capable of infecting and causing disease in humans. While the most notorious member variola, the causative agent of smallpox, was eradicated from natural infection, there are still concerns about a clandestine release during a biological attack. In addition, monkeypox and other members of the family have raised concern about epizootic infections that are capable of causing epidemic. Poxviruses produce two infectious forms, intracellular mature virus (IMV) and extracellular virus (EV). While IMV make up the majority of progeny, WV is required for efficient cell-to-cell spread and systemic infection. EV are formed by the intracellular envelopment of IMV. Their formation occurs in the cytoplasm of infected cells and only 8 proteins encoded by the virus are known to be unique to the WV form. The long-term goal of this project is to understand the molecular mechanisms employed by orthopoxoviruses to form, transport, and release infectious WV. The immediate goal of this application is to understand what regulates the transition of IMV to EV. Our hypothesis is that the virus encodes a molecular switch that gradually accumulates to sufficient quantities to block the formation of EV thus allowing the accumulation of IMV. Our specific aims are; 1) Investigation of the temporal relationship of the IMV-EV transition, 2) Perform a genetic screen for proteins involved in EV formation/inhibition. The results obtained will provide greater insight into the molecular mechanism poxviruses use to regulate the formation of the two forms of infectious virions and give us a better understanding of poxvirus morphogenesis and egress. PUBLIC HEALTH RELEVANCE: Even though natural occurring smallpox was eradicated, concern for orthopoxviruses, ranging from a clandestine release of smallpox to outbreaks of epizootic infections such as monkeypox, still exist. Understanding the biology of poxviruses is still a priority not only for preventing epidemics but also for understanding basic mechanisms of biology. The results from this study will provide new insights into poxvirus infections, which will provide new targets for antivirals and treatments against their infections.